Group photo – Participants of the TC Project RAS/5/040
To Our Readers
The IAEA provides continuous support through various regional and national technical cooperation projects to national crop improvement programs in Asia. A recently con- cluded IAEA-RCA project on Mutant Multi-location Trial and Mutational Enhance- ment of Genetic Diversity (RAS/5/040) has developed 33 mutant varieties and dozens of promising mutant breeding lines with high yield potential and/or improved agro- nomic characters, resistance, and end-user quality traits. Through the regional mutant multi-location trials (RMMT), six varieties were selected to be potentially released in countries other than where they originated. In this issue, papers are mainly contributed by participants of the RAS/5/040 project. We thank Dr. GSS Murty of Bhabha Atomic Research Centre, India (retired) for his help in editing those papers.
As planned, we have been trying to further improve the quality and widen the dissemi- nation of this publication. First, an Editorial Board has been established during the past months. Second, we are planning to rename the publication Plant Mutation Research to
Vol. 1, No. 3 December 2007
http://www-naweb.iaea.org/nafa/index.html ISSN 1011-260X
http://www.fao.org/waicent/FAOINFO/AGRICULT/Default.htm
Contents
• To Our Readers 1
• Symposium
Announcement 3
• Table of Contents 6 Included Sample Papers:
• Mutation Breeding and Genetics in
Korea 7
• Genetic
Enhancement of
Groundnut 16
• Virus Resistant
Banana 22
• Ion Beams Implantation on
Wheat 31
• Trombay Mutant Groundnut
Varieties 46
• Lodging Tolerant
Rice Variety 52
• Author’s
Guidelines 54
Last but not least, we are organizing an International Symposium on Induced Mutations in Plants, 12-15 Au- gust 2008 in Vienna, Austria, in cooperation with various organizations, e.g. Chinese Society of Agricultural Bio- technology, the European Association for Research on
Plant Breeding (EUCARPIA), Indian Society of Genet- ics, and Japan National Institute of Agrobiological Sci- ences, etc. I look forward to your participation.
Qingyao Shu
Announcements
International Symposium on “Induced Mutations in Plants (ISIM)”, Interna- tional Atomic Energy Agency, Vienna, Austria, 12-15 August 2008
Announcement and Call for Papers 1. Background
The year 2008 will mark the 80th anniversary of mutation induction in crop plants. The application of mutation techniques, i.e. gamma rays and other physical and chemical mutagens, has generated a vast amount of ge- netic variability and has played a significant role in plant breeding and genetic studies. The widespread use of in- duced mutants in plant breeding programmes throughout the world has led to the official release of more than 2600 mutant crop varieties. A large number of these varieties (including cereals, pulses, oil, root and tuber crops, and ornamentals) have been released in developing countries, resulting in enormous positive economic impacts.
The International Symposium on Induced Mutations in Plants (ISIM) will be the eighth in the Joint FAO/IAEA Programme’s Symposium series dedicated exclusively to harnessing and disseminating information on current trends in induced mutagenesis in plants, the first of which was held in 1969 and the last in 1995. These previous symposia dealt with themes relating to the development of efficient protocols for induced mutagenesis and their role in the enhancement of quality traits, as well as resis- tance to biotic and abiotic stresses in crops and the inte- gration of in vitro and molecular genetic techniques in mutation induction.
Since 1995, there has been an increased interest within the scientific community, not only in the use of induced mutations for developing improved crop varieties and for the discovery of genes controlling important traits and understanding their functions and mechanisms of actions, but also in deciphering the biological nature of DNA damage, repair and mutagenesis. A symposium that brings together the key players in basic research, as well as in the development and application of technologies relating to the efficient use of induced mutations for crop improvement and empirical genetic studies, is therefore justified and necessary.
2. Main Topics
Topics to be addressed at the symposium:
Molecular genetics and biology of physical, chemi- cal and transposon-induced mutagenesis
Induced mutations in crop breeding programmes
Mutation induction for gene discovery and func- tional genomics, including targeting induced local lesions in genomes (TILLING) and other reverse genetic strategies
Mutational analysis of important crop characters (tolerance to abiotic stresses, resistance to diseases and insects, quality and nutritional characters, etc.)
Socio-economic impact of widespread mutant varie- ties.
3. Target Audience
It is envisaged that this symposium will not only attract eminent basic research scientists but also active plant breeders from all over the world. Therefore, the sympo- sium will at once provide the platform for the exposition and rigorous discourse on current research and technol- ogy development in this field and establish linkages among scientists in order to develop knowledge-based breeding strategies and mechanisms for sharing informa- tion and resources. It will also be a venue for project managers of international and national organizations, as well as multinational and private companies engaged in plant breeding activities, to gain insights into the applica- tions of, and current trends in, mutation techniques.
4. Exhibits
Limited space will be available for commercial vendors’
displays/exhibits during the symposium. Interested par- ties should contact Mr. Qingyao Shu, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture IAEA, at e-mail: q.shu@ iaea.org.
5. Contributed Papers and Posters
Concise papers on issues falling within the topics out- lined in Section 2 above may be submitted as contribu- tions to the symposium.
(a) Submission of synopses
Persons who wish to present a paper or poster at the sym- posium must submit an extended synopsis (in English) of 800 words maximum (i.e. two A4 format pages of single spaced typing or the equivalent, including any tables or diagrams and a few pertinent references) on one of the topics listed under Section 2. The extended synopsis should be submitted together with the completed Form for Submission of a Paper/Poster (Form B), and the Par- ticipation Form (Form A) to the competent national au- thority for official transmission to the IAEA in time for them to be received by the IAEA by 17 December 2007.
Authors are urged to make use of the Synopsis Template in Word on the symposium web page (see Section 15).
The specifications and instructions for preparing the syn- opsis and how to use the synopsis template are given in the attached instructions. Also attached is a sample ex- tended synopsis.
The synopsis should give enough information on the con- tents of the proposed paper to enable the selection com- mittee to evaluate it. Introductory and general matters should not be included. The synopsis - if accepted - will be reproduced in unedited form in the Book of Extended Synopses; the original must therefore be submitted as a camera-ready copy in a form in which the author will wish to have the work presented. The general style and presentation should be as in the attached sample.
(b) Acceptance of Papers for Oral Presentation and Poster Presentation
Given the number of papers anticipated and the need to provide ample time for discussion, the number of papers that can be accepted for oral presentation is limited. Au- thors who would prefer to present their papers in a poster session are requested to indicate this preference on Form A with which they send the extended synopses.
Authors will be informed whether their papers/posters have been accepted for presentation on the basis of the extended synopsis. Guidelines for the preparation of the papers and the deadlines for their submission will be pro- vided at that time.
The IAEA reserves the right to decline to present or pub- lish any paper that does not meet expectations based on the information in the extended synopsis.
Further details about the preparation of papers and oral presentation at the symposium will be sent to the authors of the papers accepted together with notifica- tion of acceptance.
6. Expenditures
No registration fee is charged to participants.
As a general rule, the IAEA does not pay the cost of at- tendance, i.e. travel and living expenses, of participants.
However, limited funds are available to help meet the cost of attendance of selected specialists mainly from developing countries with low economic resources.
The grants awarded will be in the form of lump sums usually covering only part of the cost of attendance. Gen- erally, not more than one grant will be awarded to any one country.
If governments wish to apply for a grant on behalf of one of their specialists, they should address specific requests to the IAEA to this effect. Governments should ensure that applications for grants are submitted by 17 Decem- ber 2007 and are accompanied by a duly completed and signed Grant Application Form (as attached). Applica-
tions that do not comply with these conditions cannot be considered.
7. Symposium Proceedings
The proceedings of the meeting will be published by the IAEA as soon as possible after the symposium.
8. Distribution of Documents
A preliminary programme of the symposium will be sent to participants in advance. The final programme and the book of extended synopses will be distributed at registra- tion.
9. Working Language
The working language of the symposium will be English.
10. Participation
All persons wishing to participate in the symposium are requested to register in advance online. In addition they must send a completed Participation Form (Form A) and if relevant, the Form for the Submission of a Paper (Form B) and the Grant Application Form (Form C) through the competent official authority (Ministry of Foreign Affairs, Ministry of Agriculture, national FAO committee, or na- tional atomic energy authority) to the IAEA. A partici- pant will be accepted only if the Participation Form is transmitted through the government of a Member State of the Sponsoring Organizations or by an organization in- vited to participate.
Participants whose official submissions have been received by the IAEA will receive further informa- tion on the symposium approximately three months before the meeting. This information will also be posted on the symposium web page.
11. Accommodation
Detailed information on accommodation and other sym- posium related information will be sent to all designated participants well in advance of the symposium. This in- formation will also be available on the symposium web- site.
12. Visa
Designated participants who require a visa to enter Aus- tria (Schengen State) should submit the necessary appli- cations to the nearest diplomatic or consular representa- tive of Austria or any other consular authority of a Schengen partner State representing Austria as early as possible (please note that it could take up to three weeks to obtain a visa).
13. Channels of Communication
The Participation Form and as applicable, the Form for Submission of a Paper/Poster, and the Grant Application Form, should be sent to the competent national authority (Ministry of Foreign Affairs, Ministry of Agriculture, national FAO committee, or national atomic energy au- thority) for official transmission to the IAEA.
Subsequent correspondence on scientific matters should be sent to the Scientific Secretary and correspondence on administrative matters to the IAEA Conference Services Section.
14. Symposium Secretariat The Address of the Secretariat:
International Atomic Energy Agency IAEA-CN-167
Vienna International Centre P.O. Box 100
Wagramer Strasse 5 1400 Vienna, Austria
Tel.No.: +43 1 2600 (0) plus extension Fax No.: +43 1 26007
E-mail: official.mail@iaea.org
E-mail for paper submissions: plant.mutation@iaea.org The Scientific Secretary of the Symposium:
Mr. Qingyao SHU
Plant Breeding and Genetics Section
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture
Tel.No.: +43-1-2600 ext. 21617 E-mail: Q.Shu@iaea.org
Symposium Organizer:
Ms. Karen MORRISON Conference Services Section
Division of Conference and Document Services Tel.No.: +43-1-2600 ext. 21317
E-mail: K.Morrison@iaea.org 15. Symposium Web Page
Please visit the IAEA symposium web page regularly for new information regarding this symposium: http://www- pub.iaea.org/MTCD/Meetings/Announcements.asp?Conf ID=167
Table of Contents
Genetic Improvement of Crop Plants by Mutation Techniques in Korea
Kang, S.-Y., Kim, D.S. and Lee, G.J... 7 Genetic Enhancement of Groundnut through Gamma Ray Induced Mutagenesis
Badigannavar, A.M. and Murty, G.S.S... ...16 Selection and Characterization of Gamma Ray Induced Bunchy Top Virus Resistant Mutants in Banana
cv. Lakatan (Musa sp. AA)
Damasco, O.P., Dizon, T.O., Estrella, J.B., Caymo, L.S., Guittap, E.J., dela Cruz Jr., F.S. and Mendoza,
E.M.T... 22 Biological Effects of High Energy 7Li Ion Beams Implantation on Wheat
Guo, H.J., Liu, L.X., Han, W.B., Zhao, S.R., Zhao, L.S., Sui, L., Zhao, K., Kong, F.Q. and Wang, J.... 31 Genetic Improvement of Soybean Variety JS 80-21 through Induced Mutations
Manjaya, J.G. and Nandanwar, R.S... 36 Inter Simple Sequence Repeat (ISSR) Markers for Detecting Radiation Induced Polymorphisms and its Application as
Genetic Marker System in Sesbania rostrata (Bremek. & Obrem.)
Joshi-Saha, A. and Gopalakrishna, T...... 41 Evolution of Trombay Groundnut Varieties through Mutation and Recombination Breeding
Badigannavar, A.M., Murty, G.S.S. and Kale, D.M... 46 A Salt Tolerant Mutant Wheat Cultivar ‘H6756’
Liu, L.X., Zhao, L.S., Guo, H.J., Zhao, S.R., Wang, J., Chen, W.H. and Zheng, Q.C...... 50 A Rice Mutant Variety with Lodging Tolerance and High Yield
Kang, S.-Y., Shin, I.C., Song, H.S., Kim, D.S., Lee, G.J., Kim, J.B. and Cho, Y.C.... 52
Author’s Guidelines... 56
Review
Genetic Improvement of Crop Plants by Mutation Techniques in Korea
S.-Y. Kang1, D.S. Kim and G.J. Lee
1RadiationBreeding and Genetics Laboratory, Advanced Radiation Technology Institute, Korea Atomic Eenergy Research Institute, 1266 Shinjeong-dong, Jeongeup, Jeonbuk 580-185, Republic of Korea, e-mail: sykang@kaeri.re.kr
Abstract
In Korea, mutation breeding in crops was started in the early 1960s. Although mutation breeding method has not been used heavily for the development of plant varieties and genetic re- sources, more than 30 varieties have been released since then.
These mutant varieties released in Korea were mostly food and oilseed crops, and the major target was to improve agronomic traits such as yield, lodging tolerance, early maturity, or multi- disease resistance, etc. Additionally, horticultural crops includ- ing the rose of Sharon were improved to have a unique flower color and shape, dwarf-type height, or leaf stripes. Most of the varieties developed in the past were selected after exposing seed materials to radiation or chemical mutagens. Since the mid-1990s, the Korea Atomic Energy Research Institute (KAERI) has adopted new biotechnologies (such as tissue cul- ture, in vitro selection, doubled haploids, molecular makers, sequence analysis of genes for marker development, genomics tools, etc.) which have been integrated with traditional muta- tion breeding methods to achieve wider mutant spectra and higher selection efficiency. Currently mutant libraries of rice, soybean, and other horticultural crops are in the processes of collecting more mutants, characterizing visual and molecular features and depositing the accumulated data, which will be a valuable resource for genetic and genomic studies. Govern- ment support was provided to construct a radiation-specific research center, Advanced Radiation Technology Institute (ARTI) in KAERI, which has essential facilities for mutation breeding and irradiation. Along with the traditional breeding approaches, a mutation breeding program, driven mostly by the ARTI, is ready to play a key role in the advancement of radiation applications to a wide range of basic and applied sci- ences, including the improvement of diverse plant varieties in Korea.
Keywords: Breeding, mutant, pepper, rice, soybean Introduction
Genetic improvement of crop plants has made a major contribution to the production of food, feed, and bio- materials. Crop breeding efforts are aimed at increasing the yield and economic value by incorporating disease and insect resistance, better grain quality and a shorter growth duration. Significant achievements in the applica- tion of radiation and isotopic techniques (RT) have been made in agriculture and biotechnology (BT) fields throughout the world (Song & Kang 2003). The use of radiation induced mutations in agriculture is considered a symbol of the peaceful use of atomic energy; therefore the IAEA is promoting the use of induced mutation tech- niques in crop improvement. According to the FAO- IAEA Mutant Varieties Database (http://www-
mvd.iaea.org), by 2007 about 2600 plant varieties were released worldwide which have been derived from the RT and chemical mutagens during the past seventy years.
In Korea, mutation research in crops was begun in the early 1960s and about 30 plant cultivars have been re- leased so far. The main objective of this report is to summarize important achievements made by the applica- tion of plant mutation breeding in Korea.
Brief history of mutation breeding in Korea
In Korea, mutation breeding in crops was begun in the early 1960s and the Radiation Agriculture Research Insti- tute (RARI) was established for the peaceful use of atomic energy in the field of agriculture in 1966. From the mid 1960s to early 1970s, mutation breeding was rec- ognized as one of the newest breeding techniques in Ko- rea, and many plant breeders tried to apply the mutation techniques in their crop breeding programmes. The Rural Development Administration (RDA) and Seoul National University succeeded in the development of indica- japonica hybrid ("Tongil type" rice) cultivars with semi- dwarf and high yields by cross breeding in 1971. There- after, the main work in crop breeding was undertaken by the RDA due to the strong policy of the Korean govern- ment to increase rice production. RARI was incorporated into KAERI in 1973. As a result, most of the researchers related to mutation breeding moved to the RDA and to universities. Although the breeding research team was drastically reduced, the radiation breeding research team at KAERI played a major role in most of the mutation research in many crops. By means of mutation breeding, more than 30 new cultivars were developed; in rice: 17, sesame: 6, hibiscus: 5, soybean: 3, barley: 1, perilla and boxtron (= Chinese matrimony vine, Lycium chinensis Millor): 2 each, and distributed by KAERI singly or jointly with RDA (Table 1). Compared to other devel- oped countries, mutation techniques in Korea have not been fully used for the development of plant cultivars and genetic resources. Since the mid-1990s, KAERI's re- search team has established new radiation plant breeding technologies in combination with newer ones, such as tissue culture, in vitro selection, molecular maker and gene analysis for developing high quality cultivars. Re- cently, plant mutation techniques in Korea have been gradually highlighted again in the fields of functional genomics, as well as breeding methods.
Table 1. Mutant varieties bred in Korea Plant species Mutant cultivars
Rice Milyang-10, IRI-307, Wonpyongbyeo, Wonkwangbyeo, Wonmibybyeo, Huegseonchalbyeo, Woncheongbeyo, Wonpumbyeo, Wonchubyeo, Heugkwangchalbyeo, Nongwonchalbyeo, Seonong-6, Seonong-8, Seonong-9, Seolgaeng, Baegjinju, Goami-2
Barley Bangsa-6
Soybean KEX-2, Bangsakong, Josangseori
Sesame Ahansankkae, Suwonkkae, Yangbaeckkae, Pungsankkae, Seodunkkae, Suwon 128
Perilla Dasil, Kwangim
Hibiscus Baegseol, Ggoma, Seonnyo, Daegoang, Changhae boxtron Cheongdae, Myoengan
Main achievement of plant mutation breeding in Ko- rea
Rice
Rice is the most important crop grown in Korea in about 1 m ha and new cultivars have been bred mainly by cross breeding. The first rice mutant variety developed in Ko- rea was "Milyang 10", derived by X-ray irradiation, which had a short culm, early maturity and was disease resistant in comparison with its parent cv. "Palgwaeng".
Another mutant variety, IRI-307 was bred in collabora- tion between KAERI and the Honam Crop Experiment Station of RDA.
In 2000, KAERI released four rice mutant varieties, namely,, "Wonpyungbyeo", "Wonkwangbyeo", "Won- mibyeo" and "Heukseonchalbyeo" by irradiating with gamma rays after registering with the National Cultivar List. These new cultivars have an earlier maturity, better lodging tolerance and multi-disease resistance than their original cultivars (Shin et al. 2001 a, b, c). In 2003 an- other rice cultivar "Woncheongbyeo" was registered with the National Cultivar List. This cultivar had a short culm and high lodging tolerance and improved early maturity, compared to the parent "Chucheongbyeo", which has good taste and quality but was susceptible to lodging (Kang et al. 2006a). All of the newly developed varieties
"Wonchubyeo"(Kang et al, 2006b), "Wonpumbyeo",
"Nogwonchalbyeo", and "Huegkwangchalbyeo" were officially released to farmers in 2004. Rice seeds of the above-cited cultivars were produced by KAERI and dis- tributed directly to farmers. Further, thirty promising mu- tant lines having salt tolerance, high quality and a high yield were selected by gamma ray irradiation, and con- ducted the evaluation of their characteristics during a re- gional productive trial. Rice mutation breeding is one of the major projects at KAERI. Recently, by the treating seeds with N-methyl-N-nitroso urethane (MNU), three rice cvs. "Seonong-6", "Seonong-8" and “Seonong-9”
were released by Seoul National University and three rice cvs. "Baegjinju", "Seolgaeng", Goami-2 by National Crop Experiment Station (NCES) of the RDA (Table 1).
Soybean
Soybean is one of the most important crops on the Korea peninsula, where it has been known as an original genetic
center for leguminous crops (Hymowitz, 2004). The soy- bean variety "Kwanggyo" was first released by cross breeding in 1969. At the same time, mutation breeding on soybean was started at the RARI and RDA. "KEX-2" was released in 1973 as a first mutant cv. It was developed after X-ray irradiation to "Kuemgangdaelip", which was a leading cv. in the 1970s, but had late maturity. "KEX- 2" matured 11 days earlier than parent cv. and fitted well into the barley-soybean cropping system in Korea. Fur- ther, a new soybean cv., "Bangsakong" was developed by a joint work between the RDA and KAERI in 1983.
When seeds of "CB-27," introduced from the USA were irradiated by 250 Gy gamma rays, "Bangsakong" was selected. It was recommended as a suitable cv. for the growing of a soy sprout with a small seed size 100-seed weight (HSW) 12g. Besides, "Bangsakong" showed higher yield (2.05 t/ha) compared to the local cvs. (1.53 t/ha) by which it was broadly cultivated in Korea (Lee, 1997).
Although mutation breeding for the soybean resulted in only two cvs., "KEX-2" and "Bangsakong" during >30 years in Korea, continuous research on the soybean has been conducted in various fields for breeding purposes using gamma ray irradiation to the seeds at KAERI. At the end of 2006 from the M8-M12 generations, several promising lines were selected from the gamma ray mu- tated progenies, such as 6 lines with large seed size and resistance to pod and stem blight, 73 lines with small seed and higher yield, and 16 lines for cooking purpose.
Besides, 16 lines with a low lipoxigenase (LX-1, LX-2, LX-3) content and 6 lines with a 20% lower phytic acid content than the parent were selected. These lines are cur- rently subjected to confirmation at the biochemical and molecular levels before submitting for a plant variety protection in Korea. A new line for cooking with rice was selected, tested, and now applied for a plant variety pro- tection right in 2005. The black seed-coated variety
‘Josaengseori’ had a smaller seed size (32.8 g HSW) and an early maturing (~30 days earlier) characteristic when compared to the original ‘Seoritae’ which had 40.1 g HSW. At KAERI, about 4,000 varieties/lines of native leguminous resources were collected and continuously proliferated, preserved and evaluated (Choi et al. 1999).
Mutation research by chemical mutagens in soybean has also been conducted, especially to develop root traits
such as greater nodulation by the Seoul National Univer- sity and NCES-RDA.As a result, supernodulating mu- tants were selected on using EMS, which were character- ized by a smaller plant height, greater dry nodule weight and number/plant than the parent, "Sinpaldalkong 2"
(Lee, 1997).
Oil crops: sesame and perilla
Both sesame (Sesamum indicum L.) and perilla (Perilla frutescens Briton, Syn. to Perilla. ocymoiides L.) oil are widely used for cooking. The leaves of perilla are also commonly used as a vegetable. Mutation breeding of ses- ame has been mainly conducted at the NCES of RDA since the late 1970s. First a sesame mutant cv., "Ahn- sankkae", was successfully developed after irradiating with 200 Gy X-rays to the seeds of a local cv., "90 days chamkkae". The mutant has desirable characteristics such as pure white seed coat, resistance to diseases, disasters and higher yield. Therefore, it was rapidly grown in more than 30% of total sesame cultivated area, 50,000 ha for a while in the late 1980s (Kang, 1997). "Ahnsankkae",
"Suwon-128", "Yangbaeckkae", and "Seodnkkae" were developed following chemical mutagen, sodium azide (NaN3) 2mM for 2.5h treatment to the seeds at NCES.
"Suwon-131" and "Pungsankkae" were bred by crossing mutant DR-45 with other cultivars.
Mutation breeding of perilla was initiated at NCES in 1990. Gamma rays with 150-400 Gy and 0.5% EMS were applied to the dry perilla seeds of cvs. "Yechun", "Suwon 38," Yeupsildlkkae and Okdongdlkkae. In the M2, 1,875 variants were selected (Park, 1997). In 2003, two new varieties of perilla were released. "Dasil" with early ma- turity and dwarf habit, was derived on irradiating with 350 Gy gamma rays to the seeds of "Yechun" (Park et al., 2003a). "Kwangim" with a high yield and leaf rust resis- tance, was induced by EMS treatment to Suwon 38 seeds (Park et al., 2003b).
In order to breed new leaf edible varieties, perilla cv.,
"Chukyopzaso" was irradiated with 200-350Gy gamma rays and various mutants affecting leaf shape, chlorophyll and anthocyanin were isolated in the M2-M3 generations.
The chlorophyll content in perilla leaves ranged from 52% to 134%, compared to the parent. It had increased in 14 mutant lines and decreased in 15 lines. The anthocya- nin content ranged from 74% to 157% and increased in 26 mutant lines and decreased in only 5 lines (Lee et al., 1999a). "Chukyopzaso" also gave rise to different vari- ants in leaf flavor components. Promising mutants with high contents of limonene were obtained from 8 lines, perillaldehyde from 8 and α-pinene from one line (Lee et al., 1999b). The promising lines are under field trials for the characterization of their agronomic traits and analysis of the functional compound to register as a new variety.
Hibiscus (Rose of Sharon)
and other countries. The first induced mutant developed was "Baekseol" (meaning 'white snow'). It was derived from a bud mutation by a 50 Gy gamma ray irradiation to the cuttings of "Hwarang" (Song et al. 1999). Until now, four mutants were officially registered as new varieties and then released - "Seonnyeo" and "Daegoang" in 2003 and "Ggoma" and Changhae in 2006. "Seonnyeo" has light purple petals and white core lines on the inner pet- als. It was selected from a mutant line irradiated with gamma rays (100 Gy) to the seeds of "Gyewolhyang"
which had dark purple petals and red core lines (Song et al, 2005a). "Daegoang", with a large flower size (12-14 cm in diameter) was derived from the mutant of
"Younggwang" (8-10cm in flower diameter) (Song et al.
2002b). "Ggoma" (meaning of 'kid') is a dwarf mutant selected from the 100 Gy gamma ray irradiated seeds of
"Hongdansim-2" (Song et al. 2005b). Besides shorter plant height, flower and leaf size are smaller than parent cv. In an 8-year-old shrub, the height and flower diameter are 1m and 5cm for "Ggoma" and 2.5m and 9-10cm for
"Hongdansim-2", respectively. "Ggoma", can be grown as a 'bonsai' or indoor cultivation (Song et al, 2006a).
Changhae was mutated from original cultivar Sumino- kura and shows a bluish purple color and a large flower size (Song et al., 2006b). New mutant lines of hibiscus with various plant and flower types were selected and their characteristics are continuously being evaluated.
Flowers
Since the 1990s, breeding of flowers has been empha- sized in Korea. The National Horticulture Research Insti- tute (NHRI) of the RDA, applied gamma radiation of 10~50 Gy to the young plants of Chrysanthemum propa- gated by tissue culture. Various mutants with a changed flower color and shape were obtained in the M1V3 gen- eration, namely, "Bongwhang", "Herman De Boon", "Hi- jeong" and "Paso Doble". Pink flower color changed to yellow, orange, bronze and red. In some of the mutants, purple color changed to white, pink and red, and white changed to yellow and pink. The selected promising lines were evaluated for their characteristics (Goo, 2000). A wide range of plant materials were subjected to exposure to gamma rays for mutation induction in chrysanthemum in Korea, which include cuttings, rooted plants, and calli derived from petals or stems. Stem segments of a spray- type chrysanthemum (cv. Argus) were cultured on MS medium containing 1.0 mg•L-1 NAA and 1.0 mg•L-1 ki- netin, and the regenerated plantlets on NAA and BA combination medium were treated with various doses of gamma rays. Results indicated that gamma ray radiation ranging from 30 to 50 Gy was effective in in vitro mutagenesis for the flower color, flower shape, and stem color of the spray-type chrysanthemum. In 50 Gy of gamma-ray irradiation, 28.2 and 15.4 % of the individu- als varied from the original variety in the flower color and shape, respectively (Park et al., 2007). Development
farmers due to its labor saving culturing and economic gains.
Besides the Chrysanthemun, at present many ornamental plants including rose, Rhododendron, lily, lawn grass, and native wild flowers are being used for mutation breeding at many national and provincial agriculture re- search institutes and universities. KAERI has also just started the mutation breeding of floricultural and native plants to develop new plants with desirable traits. An- other increasing market in potting flower is the orchid.
With collaboration with the private sector, KAERI re- cently released novel mutants through combining use of technologies of tissue culture and gamma irradiation. One of the new varieties, "Dongi", was derived from the im- ported oriental orchid Cymbidium. Another variety "Eun- seol" was originated from the Korean indigenous orchid
"Sokgok". Both varieties were favored for their fine and unique stripes along the leaf edge.
Pepper
In collaboration with KAERI, Nongwoo Bio Co. Ltd., one of the major Korean seed companies developed the disease tolerant lines of hot pepper (Capsicum annuum L) by using radiation and interspecific hybridization. Many variants were selected by irradiation with 250Gy, but most plants were recessive types and were found to be inferior. However, 12 lines from the M6 generation were selected based on the fruit shape and fruit setting ability.
Sixteen lines from the M6 by pedigree breeding method resulted in the selection of the 95240 line, which is resis- tant to phytophthora root rot. This line showed diverse phenotypes and can be used as resistant sources for the phytophthora root rot, once they become bred-true.
Current trends in using mutation techniques with the RT and BT combination techniques
Application of a nuclear technique for a solution to the food problem has been useful in providing an opportunity to accelerate plant improvement. Current research pro- grams emphasize on the development of techniques of plant tissue culture and on the induction and selection of radiation mutations. Since the mid-1990s, at KAERI new radiation plant breeding technologies have been estab- lished to breed cultivars with resistance to environmental stresses and a high quality in combination with new bio- technologies such as application of tissue culture, in vitro selection, doubled haploids, molecular maker, and gene analysis, etc.
Development of NaCl tolerant rice plants through the irradiation of gamma rays and tissue culture
Selection and regeneration of NaCl tolerant cell lines Callis were treated with 30~90 Gy gamma rays on a me- dium containing 1.5% NaCl to obtain the NaCl tolerant cell lines. The frequency of regeneration in the irradiated (30Gy and 50Gy) calli was higher than that of the unirra- diated, but decreased at over 70 Gy. Proline, phenolic compounds, sugar and protein contents in tolerant calli were 0.6, 10, 5 and 4 times more than those of non-
selected ones, respectively and the peroxidase activity was 2 times more. Proline content of the plantlets derived from tolerant callus was 3 times higher than that of con- trol. Leaves of the regenerants (M1) derived from the salt tolerant calli were not normal in their shape compared to those of the corresponding "Dongjinbyeo" because of their frequent mutation (Lee et al., 2002a).
Double haploids have long been recognized as a valuable tool in plant breeding, since they not only offer the quickest method of advancing the heterozygous breeding lines to homozygosity, but also offer an increased selec- tion efficiency over conventional procedures due to a bet- ter discrimination between the genotypes within any one generation. Salt tolerant mutants were obtained in the rice cv. Hwaseongbyeo, through in vitro mutagenesis of an- ther cultured calli. Various doses (30, 50, 70 and 90 Gy) of gamma rays were used to investigate the effect of ra- diation on the callus formation on a medium containing 1% NaCl, green plant regeneration, frequency of the se- lected double haploids and salt tolerance screening. It was demonstrated that 30 and 50 Gy gamma rays have a significant effect on the callus formation, regeneration and the selection of salt tolerance. No tolerant line was obtained from unirradiated cultures. From gamma ray irradiated cultures, five lines in the M2 and 13 lines in the M3 showed tolerance at germination and seedling stages, respectively. The frequency of the salt tolerant mutants indicates that anther culture in combination with gamma rays is an effective way to improve salt tolerance (Lee et al., 2003a).
Selection of salt tolerant lines at M2 and M3
To investigate the germination rate, seeds (M2) were kept in a solution of 1.5% NaCl for 10 days. The germination rates of the M2 lines ranged from 0 to 50% and the toler- ant lines with a superior ability in growth were screened.
The ratio of segregation in the tolerant line vs the sensi- tivity line was 1:1. The salt tolerant lines (M3) at the seedling stage were isolated and compared to the Dong- jinbyeo. The ATPase, catalase and peroxidase activity of the tolerant lines were increased in the mutants. But, the electrical conductivity, starch and Na+ content were de- creased in mutants compared to the Dongjinbyeo (Lee et al., 2003 d).
Selection and test of tolerant lines in a saline field
Among the planted lines in the saline field, survival rate, plant height, panicle length, no. of hills and fertility rate of lines nos. 18, 50 and 268 had increased compared to Dongjinbyeo. Salt tolerant lines, harvested in the year 2001 were again sown at the saline field (Dobido) with two replications and designated as salt tolerant because of the increased biomass and grain yield (Lee et al., 2003b).
DNA and protein analysis of the salt tolerant lines
AFLP and RAPD techniques were used to classify the tolerant and sensitive lines. Specific bands derived from the E/ACC + M/CTG combination primer in the AFLP and the F-08 primer in the RAPD were observed in toler-
ant lines. The AFLP and RAPD bands were sequenced and analyzed for nucleotide sequence homology. AFLP band has homology with rbcL gene and cold stress - in- duced cDNA (Lee et al., 2003c).
The translation products were analyzed by two- dimensional gel electrophoresis. About 300 polypeptide spots appeared in plant leaves, 6 spots showed in only the tolerant lines and many spots showed an increase, in a relative amount. 11 polypeptides were isolated and se- quenced. Among the 11 polypeptides, only 4 polypep- tides could be sequenced. The homology search revealed that two peptides of 100-kD had a significant homology with phosphoribulokinase, a 35-kD protein with an oxy- gen evolving enhancer 1 and a 25-kD protein with a H+- ATPase (Lee et al., 2004).
Characterization of 5-methyltryprophan (5MT) resistant rice mutants with high amino acids
Selection of 5MT resistant mutants
Rice is one of the most important crops for the human dietary system. However, rice which forms the staple food of a majority for the people is very low in trypto- phan and lysine in the storage proteins within the en- dosperm. Therefore, it is a matter of concern and also interest to know about nutritional problems in a country which lives on rice, whether there is sufficient intake of essential amino acids through rice, since they are not syn- thesized in humans and monogastric animals. For im- provement of the grain quality in rice, 5MT resistant cell lines were selected by in vitro mutagenesis using gamma rays. 5MT resistance was expressed in the regenerated plants and their progenies. Two lines, MRI and MRII, were obtained from the regenerated plants of these 5MT resistant cell lines. Both lines were successively propa- gated by selfing, which resulted in four homozygous lines in the M3. Protein content of brown rice was increased by about 19% and 32%, and the total content of free essen- tial amino acids increased to 71% and 34% over control in MRI and MRII groups, respectively.
Development of AFLP and STS markers
To develop a genetic marker for the identification of the 5MT resistant mutants in early generation, AFLP analy- sis was conducted with the control, homozygous MR lines, and segregating lines. Of the 3684 AFLP bands from the 45 primer combinations by eight EcoRI(+2) and MseI(+3) primers, 361 (9.8%) were polymorphic in the 5MT resistant mutants. The size of the polymorphic fragments ranged from 55 to 313 bp in length. Mutants were grouped into three clusters in a cluster analysis through the UPGMA method. Ten out of the 36 se- quenced polymorphic PCR products were used for de- signing the primer sets for STS analysis. In the test with two homozygous M4 5MT resistant mutants, six STS primer sets (OSMR1, OSMR2, OSMR3, OSMR4,
control and the seven M2 progenies phenotypically resis- tant to 5MT (Kim et al., 2004 b). These STS markers can serve as potentially 5MT resistance-specific markers.
Expressed sequence tag (EST) analysis
To study the gene expression in the plant vegetative tis- sues, a cDNA library was constructed by using the leaves and roots of the 5MT resistant mutant plants. Comple- mentary DNA was constructed from leaves and roots of the 5MT resistant plants. Titter analysis of the secondary library indicated about a 3.55×1010 pfu/ml. Inserted DNA sizes were distributed from 0.35~2.5 kbp with the aver- age value of 1.14 kbp. The expressed sequenced tags (ESTs) of 1,019 randomly selected clones evaluated by assembling 588 non-overlapping sequences. Through BLASTx search analysis against the NCBI database, 389 unigenes with significant homologies with known protein sequences and the remaining 199 unigenes were desig- nated unidentified genes. These ESTs were grouped into 13 categories according to their putative functions. A to- tal of 126 unigenes were considered to be genes regulated by 5MT through inference from known pathways and mechanisms related to stresses or to amino acid synthesis.
Many genes that were identified tended to be related to defense and stress responses, suggesting “cross-talking”
between biotic/abiotic stresses including the 5MT treat- ment. Therefore, 5MT resistant mutants might be of value for identifying genes related to plant defenses and stresses (Kim et al., 2007a). Further work is required for identification of the physiological response and the resis- tance mechanism of a plant in response to an amino acid analog related to the other stresses.
Characterization of the altered anthranilate synthase in 5-methyltryptophan resistant rice
In order to identify the 5MT resistant mechanism, the current study further investigated these mutant lines; the anthranilate synthase activity of these M5 advanced lines was measured by a direct fluorometric detection of the formed anthranilate in the control plants and the mutant lines grown on 500 µM 5MT. The anthranilate synthase activity of the mutant plants was 2.2 - 3 times higher than that of the control. In a kinetic analysis with tryptophan, anthranilate synthase of the mutant lines was insensitive to feedback inhibition. These lines showed an enhanced accumulation of storage proteins and amino acids. The increasing rates of protein in the mutant lines, relative to the control seeds, were 17 ~ 28.5% (Kim et al. 2004).
The amino acid contents were 2.4 (MRI-40-2) ~ 2.6 (MRI-110-6) times higher in the MRI lines than those in the control seeds, and 2.4 (MRII-12-5) ~ 3.5 (MRII-8-1) times higher in the MRII lines than the control seeds.
Significant increases among the amino acids of the MR lines were observed in tryptophan, phenylalanine and tyrosine, which had been biosynthesized through the shikimate pathway. The transcript levels of putative
and other abiotic stresses (ABA, NaCl, and cold). Puta- tive OASA2 gene in the 5MT resistant mutant lines was highly expressed in a low 5MT concentration and at an early stage of the 5MT and αMT treatments. However, the mRNA accumulation of putative OASA2 gene in the mutant plants was gradually decreased by abiotic stresses such as NaCl and a cold-treatment. These results indicate that the 5MT resistance in the mutant lines was due to the altered anthranilate synthase forms (Kim et al. 2005b).
Proteomic analysis
SDS-PAGE analysis identified changes in total proteins and purified protein fractions based on their solubility properties between the control and the mutant lines. The 5MT can fit into the allosteric site of AS in the same way as tryptophan. In conclusion, it may be an inhibition re- agent in a plant cell. This cytotoxic effect by 5MT may induce stresses in vivo similar to other environmental stresses. Proteins produced in elevated amounts or de novo in response to 5MT were studied by comparing the silver-stained two-dimensional gels of leaf proteins be- tween the control and two 5MT resistant mutant lines. At least twenty proteins were produced in elevated levels or de novo following an exposure to the growth inhibitory concentrations of 5MT in MRI-40. During the investiga- tion of the 5MT stress-mediated responses of the four antioxidant enzymes, catalase (CAT, EC 1.11.1.6), per- oxidasse (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1) and aspartate peroxidase (APX, EC 1.11.1.11), the activity levels of the four enzymes were increased by the 5MT treatment in the control and the 5MT resistant mutant lines. However, the mutant lines revealed higher increases of the antioxidant enzymes than the control. Differences of a significant activity increases between the control and the mutant lines were observed in the SOD and APX activity assays. Native PAGE con- firmed these differences in the SOD and APX activities with the separation patterns of the isoforms of SOD and APX (Kim et al. 2005a).
Selection of azetidine-2-carboxylic acid (AZCA) resis- tant cell lines by in vitro mutagenesis in rice
Resistant cell lines to the AZCA were selected through rice embryo culture after callus was irradiated with 30, 50, 70, 90 and 120 Gy gamma rays. The optimum AZCA concentration for the selection of resistant cell lines was 3 or 4 mM AZCA considering the LD50 and the fresh weight of the callus. Survival rate of the AZCA resistant callus showed a remarkable increase in the callus irradi- ated with 50 and 70 Gy. Based on the fresh weight, sur- vival rate and regeneration for the selection of the AZCA resistant cell line, 50-90 Gy was considered as the opti- mum dose range for gamma irradiation. Irradiated calli selected from AZCA were more tolerant to NaCl than those from the non-irradiated calli. This suggests that the elevated resistance to osmotic stress resulted from the mutagenic treatment. The level of the free proline content in the AZCA resistant cell line had increased up to 3.5 times compared to control. Proline content in the regen- erant derived from the AZCA resistant cell line also in-
creased to 1.7 times that from the control plants regener- ated from the callus grown in the AZCA free medium.
Selection of the proline overproducing cell lines by in vitro mutagenesis was successful and seems to be useful for the improvement of stress tolerance in rice. The se- lected lines were planted in a paddy field to analyze the growth characteristics (Hyun et al., 2003, Lee et al., 2002b).
Selected AZCA resistant lines that had a high proline accumulation were used as sources for the selection of NaCl resistant lines. To determine an optimum concen- tration for selection of NaCl resistant lines, Donganbyeo seeds were initially cultured on a media containing vari- ous NaCl concentrations (0 to 2.5%) for 40 days, and a 1.5% NaCl concentration was determined as the optimum concentration. One hundred and sixteen salt-tolerant (ST) lines were selected from 20,000 seeds of the AZCA resis- tant M3 seeds in the medium containing 1.5% NaCl. The 33 putative lines (M4 generation) considered to have a salt-tolerance were further analyzed for their salt toler- ance, amino acid and ion contents, and the expression patterns of the salt tolerance-related genes. Out of the 33 lines, 7 lines were confirmed to have a superior salt toler- ance. Based on a growth comparison of the entries, the selected mutant lines exhibited a greater shoot length by 1.5 times, root length by 1.3 times, root numbers by 1.1 times, and fresh weight by 1.5 times that of the control.
Proline contents were increased at a maximum by 20%, 100% and 20% in the leaf, seed and callus, respectively, of the selected lines. Compared to the control, amino acid contents of the mutants were 24 to 29%, 49 to 143% and 32 to 60% higher in the leaf, seed and callus, respec- tively. The ratios of Na+/K+ for most of the ST-lines were lower than that of the control, ranging from 1.0 to 3.8 for the leaf and 11.5 to 28.5 for the root, while the control had 3.5 and 32.9 in the leaf and root, respectively. The transcription patterns for the P5CS and NHX1 genes ob- served by RT-PCR analysis indicated that these genes were actively expressed under a salt stress. The selected mutants will be useful for the development of rice culti- var resistant to a salt stress (Song et al., 2007).
Development of herbicide-resistant cell lines using gamma ray irradiation
In joint research with KAERI, Suncheon University and Korea University teams have been trying to select cell lines with herbicide tolerance by radiation and in vitro culture methods in rice and wheat. Herbicide tolerant rice (Oryza sativa L. cv. Ilpumbyeo) cell lines were selected from γ-ray-irradiated anther-derived cell cultures. One line (CHB-1) showed a stable tolerance at a 10 mg/l con- centration after a 6-month culture without a herbicide suspension. These results reveal that somatic hybrids were successfully obtained by fusing the cyhalofop butyl resistant cell line with a different cultivar having plant regeneration ability.
To identify the molecular markers for bentazone resistant wheat lines, wheat mutants were generated from a culti- var “Geumgangmil” by using gamma-ray irradiation.
Bentazone resistant M2 plants were selected from seed- lings and adult plants consecutively. The Bentazone re- sistant wheat plants were able to survive at up to 10 times (1,600ppm) the commercially recommended concentra- tion. The four plants with the highest bentazone resis- tance were selected. An AFLP analysis was done to iden- tify molecular markers for bentazone-resistant wheat mu- tants. Twelve polymorphic products were identified, cloned, and sequenced. Among the twelve polymorphic bands, eight Sequence-tagged Sites (STS) primer sets were designed. Only one STS primer set (HRMW-08) was converted into a “Bentazone-resistant wheat- specific” STS marker. The wheat mutants and makers could be employed in a cross-breedeing program incorpo- rated with maker assisted selection in the early stage of population (Kim et al. 2007b).
Conclusion
The mutation breeding program in Korea has traditionally focused on major food and oilseed crops. Currently the high yield potential of the cultivated crop cultivars in the Korean field is relatively less popular than before, but the expectation of more value-added crops from the farmer’s side is in high demand. Accordingly, the mutation breed- ing program in Korea has assigned more resources to other crop species, including some floral plants, medici- nal plants, and industrial crops.
At present, besides the importance for mutation breeding, mutation techniques have been used in many germplasm enhancement programmes for functional genomics. For the use of gene tagging, some Korean research teams are producing many thousands of insertional mutant lines by T-DNA (Jeong, 2002) and transposon techniques (Han et al. 2003) in rice, Arabidopsis and others. However, inser- tional mutation techniques are very laborious and cost intensive, whereas radiation and chemical mutagenesis might be more efficient when they are combined with microarrays and other new molecular techniques such as TILLING (Targeting Induced Local Lesions IN Ge- nomes) (Perry et al. 2003). Accordingly, the development of new crop cultivars and genetic resources is of great importance, not only for crop production to satisfy the increasing food demand, but also for functional genomic research in the post-genomic era.
In the early 2000s, the Korean government started to support fundamental and applicable research in radiation fusion technologies with BT, IT (information technol- ogy), NT (nano technology), and ST (space technology).
Also, the outcome of the technologies including the agri- cultural varieties and gene information were eventually transferred to the related industries for a quick commer- cialization. The contemporary financial assistance was provided to develop essential facilities within the Ad- vanced Radiation Technology Institute (ARTI), which include research laboratories, office buildings, irradiation
gamma-phytotron, and a seed storage facility with low temperature. An electron beam irradiator, a cyclotron for the radioisotope production, and a training center will be operational shortly. The advanced facilities and compiled data at ARTI will significantly contribute to a radiation mutation breeding for international communities. Addi- tionally it is expected that ARTI, with its diverse re- sources and governmental support can play a key role in the advancement of radiation applications for a wide range of basic and applied sciences, including the im- provement of diverse plant varieties.
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Research Article
Genetic Enhancement of Groundnut through Gamma Ray Induced Mutagenesis
A.M. Badigannavar1 and G.S.S. Murty2
1Nuclear Agriculture and Biotechnology Division,Bhabha Atomic Research Centre, Trombay, Mumbai – 400 085, India, e-mail:
anandgnut@gmail.com, 2116/3392 Pantnagar, Ghatkopar (East), Mumbai – 400 07, India Abstract
Induced mutagenesis along with recombination breeding played a vital role in the genetic improvement of groundnut (Arachis hypogaea L.) at Bhabha Atomic Research Centre, India. In continuation of ongoing mutation research since the 1960s, popular cv. TAG 24 was irradiated with gamma rays in order to further generate genetic variability. As expected, higher doses of gamma rays drastically affected the seedling traits in the M1 generation. In the M2, in all 71 true breeding macro mutants affecting plant height, leaf colour, leaf type, leaf size and leaf shape, flower colour, pod type, seed size and seed colour were isolated, characterized and maintained.
Key words: Arachis, groundnut, gamma rays, mutants, herita- bility
Introduction
Cultivated groundnut exhibited a narrow genetic base despite having extensive morphological, physiological and agronomical variability. The scarcity of genetic vari- ability makes groundnut vulnerable to a wide variety of biotic and abiotic stresses. For instance, groundnut culti- vars grown in the Southern USA are highly susceptible to Meloidogyne arenaria and crop damage in heavily in- fested fields can be devastating (Nelson et al. 1989). In- duced mutagenesis was successfully applied along with cross breeding, generating a wide spectrum of variability in groundnut affecting various traits (Gregory, 1955;
Patil, 1966; Chandramouli et al., 1989; Gowda et al., 1996). Consequently, 25 mutant or mutant derivative va- rieties were developed and released for commercial culti- vation for the benefit of Indian farmers between 1970 and 2004 (Murty et al., 2004).
A blend of mutation and recombination breeding played a significant role in the development of unique traits as seen in the development of cv. TAG 24, which has a ge- nomic blend of five different mutants brought under the background of cv. M 13 (Patil et al., 1995). It has most of the ideal morpho-physiological traits, such as semi-dwarf habit, small, thick, dark-green leaves, determinate flower- ing, early maturity and enhanced dry matter partitioning (Badigannavar et al., 2002). Due to its superior perform- ance across the country, TAG 24 is used as National Check Variety in Rabi (post rainy)/summer situations in the All India Coordinated Varietal Trials. In view of its worthiness, TAG 24 was chosen for gamma ray mutagenesis to induce further genetic variability.
Materials and methods
Dry seeds of TAG 24 were irradiated with 100, 200, 300, 400 and 500 Gy of gamma rays from a 60Co source at the Bhabha Atomic Research Centre (BARC), Mumbai. In each dose, three replications with 20 seeds each along
with un-irradiated control were germinated in laboratory on filter papers kept inside Petri plates moistened with tap water. After three days, germinated seeds were trans- ferred to wooden boxes containing soil and sand (2:1) and kept under 24 h illumination at 22 ± 1°C. Observa- tions were recorded on seedling height, hypocotyl length, root length, number of leaves, number of branches and germination percentage at 15 days after sowing (DAS).
For the mutation induction study, 500 seeds each of TAG 24 were treated with 150, 250 and 350 Gy gamma rays and sown in the field (M1) along with 100 un-irradiated seeds. M2 generation was raised as M1 plant progenies.
All throughout the crop season, M2 plants were screened, variant plants marked at regular intervals, harvested indi- vidually and advanced to the M3 to study their breeding behaviour. True breeding mutants were advanced to sub- sequent generations by growing alternately in rainy (June – October) and summer (January – May) seasons till M8
generation with spacing of 50 X 15 cm and 30 X 10 cm, respectively. Pod beak, constriction and reticulation were recorded as per the groundnut descriptors (IBPGR and ICRISAT 1992).
Genotypic variability (VG), genotypic coefficient of variation (GCV), heritability in broad sense and the ge- netic advance (GA) for plant height, number of branches, pod yield (g plant-1), seed yield (g plant-1), shelling out- turn (%), 100 seed weight (HSW, g), oil content (%) and oil yield (g plant-1) were studied from the data on five randomly selected plants in two replications among 71 mutants and parent TAG 24 from M5 to M8 generations.
Results and discussion
Effect of gamma ray irradiation
With an increase in dosage of gamma rays, seedling traits were affected severely (Table 1). The reduction was more pronounced in seedling height and number of branches (Fig. 1A). Based on these observations, 150, 250 and 350 Gy doses were identified as effective doses for field ex- perimentation. In the field, the highest germination (77%) was observed in 150 Gy, while the lowest (63%) was in 350 Gy as against un-irradiated control (75%). In M1
generation, there was a reduction in the number of sur- viving plants at harvest with an increase in dosage i.e., from 68% to 30% in 150 and 350 Gy, respectively.
Induction of mutants for morphological traits
M2 generation was comprised of 785 families with a total plant population of 11,441 surviving plants at harvest. In all, 71 mutants were induced with a frequency of 0.62%
affecting various characteristics which bred true in the M3 and subsequent generations (Table 2). They were des-
ignated as Trombay groundnut mutants (TGM)s. Out of 71 mutants isolated, one mutant was obtained spontane- ously from another mutant, 28 were from 150 Gy, 36 from 250 Gy and six from 350 Gy with a mutation fre-
quency of 0.42%, 0.88% and 0.75%, respectively based on surviving M2 population per dose. This indicated that 250 Gy appeared more effective in this study.
Table 1. Effect of gamma ray irradiation on germination % and seedling (15 days old) traits Gamma ray
treatment (Gy)
Germination (%)
Root length (cm)
Hypocotyl length
(cm)
Number of leaves
Number of branches
Seedling height
(cm)
0 97.7 18.7 2.2 6.0 1.5 13.8
100 95.5 18.4 2.0 6.0 1.6 15.6
200 90.9 17.9 1.8 5.5 0.7 * 13.9
300 95.5 16.9 1.8 5.5 0.5 * 8.9 *
400 88.6 15.0 * 1.7 5.0 * 0.1 * 6.5 *
500 84.1 7.2 * 1.7 3.7 * 0.0 * 3.2 *
CD 5% 2.0 NS 0.6 0.5 2.3
* Significantly different from 0 Gy at P = 0.05
Figure 1. A: Effect of gamma rays on M1 seedlings. Left to right: Parent, 100, 200, 300, 400 & 500Gy gamma ray treated B: Parent (left) and dwarf mutant (right)
C: Parent (left) and tall mutant (right)
D: Parent (left) and TGM 36 for plant height segregation in the M3 (left to right) for Phenotypes of parent, dwarf and extreme dwarf E: Disease lesion mimic leaf mutant (left) and parent (right)
F: Virescent leaf mutant
G: Golden yellow coloured leaf mutant
H: Parent (left) and funnel shaped apical leaflets in TGM 51 (right) I: Parent (left) and dumb-bell shaped pods in TGM 59 (right)
D E
F
H I G
A B C
D E
F
H I G
D E
F
D E
F
H I G
H I G
A B C
